HIV genomes are highly variable and have been classified into nine subtypes for most prevalent group M viruses. The genetic divergence among the subtypes can be up to 30%. This high level of genetic variation has become a major hurdle for AIDS vaccine development. To overcome this problem, we have generated a group M consensus (con/con) env gene for all subtypes. The genetic distance between this con/con env gene and any subtype env gene is about the half of that between different subtypes. Western blot and ELISA assays show that the con/con Env protein can bind to the sCD, mAbs and patient sera. The Biacore assay shows that it not only can bind to the neutralizing antibodies and non-neutralizing antibodies but also can undergo conformational changes to bind to 17b mAb as native gp120s. Our preliminary results show that the con/con Env protein expresses linear, discontinuous and glycan antibody epitopes, and it contains cross-reactive epitopes for serum antibodies. Theoretical prediction shows that all well-characterized T cell epitopes and their cleavage sites are preserved on this protein. This data suggests that antibody and CTL epitopes on the con/con Env protein can be processed and presented to the immune system. Therefore, the con/con Env protein may serve as a universal immunogen for all HIV-1 subtypes. In the current proposal we will determine whether such artificially derived proteins can induce broader immune responses than subtype-specific Env immunogens by measuring both cross-clade neutralization activity and cellular immune responses in guinea pigs and mice. The animals will be immunized with naked DNA and then boosted with either rgp120 or recombinant vaccinia viruses (rVV) expressing Env proteins. We will collect serum samples from guinea pigs to determine if higher titers of cross-clade neutralizing antibodies will be elicited by the con/con Env protein, and if humoral immune responses can be further augmented by either rgp120 or rVV. We will also isolate the splenocytes from immunized mice to determine if the con/con Env protein can induce strong cross-clade cellular immune responses alone or with rVV boost by comparing to subtype specific Env immunogens. Results from this study will determine if con/con Env protein can serve as a universal immunogen for all HIV-1 subtypes and will be used as a guideline to design broadly reactive immunization strategies.